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PubMed2006

Coxiella burnetii and milk pasteurization: an early application of the precautionary principle?

Stringency of milk pasteurization has been established on requirements for Coxiella burnetii as being the most heat-resistant organisms of public heath significance. This paper discusses the estimation of the efficiency of pasteurization time/temperature combinations as required in regulations for food safety. Epidemiological studies have been interpreted as C. burnetii being a significant pathogen causing clinical disease through ingestion of milk. The paper examines the evidence and challenges the designation of C. burnetii as a foodborne pathogen. Consequently it questions the need for pasteurization parameters to be established on its heat resistance characteristics.

Animals↗
PubMed1993

Polymorphism in DNA restriction patterns of Coxiella burnetii isolates investigated by pulsed field gel electrophoresis and image analysis.

Pulsed field gel electrophoresis was used for differentiation of Coxiella burnetii isolates derived from animals in Europe and one from Namibia. Previously published data on isolates from North America demonstrated four groups with distinct banding patterns. Fingerprints of prototype isolates as references confirmed four different cleavage patterns upon NotI restriction of total DNA as described. With isolates available at our institute five additional DNA banding patterns were obtained from European and one Namibian isolate after endonuclease restriction with NotI. To compare patterns from different electrophoretic runs more precisely an image analysis system was used.

Animals↗
PubMed1992

Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli.

A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.

Amino Acid Sequence↗
PubMed1994

Immunogenicity of Coxiella burnetii whole cells and their outer membrane components.

The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of protection against infectious Cb I was demonstrated after immunization with TCAE, PRO, and LPS I. Other OMC did not induce protection against this agent.

Animals↗
PubMedDate not supplied

Entry of Coxiella burnetii into host cells.

The attachment to and entry into L mouse fibroblast cells of viable phase I and phase II Nine Mile Coxiella burnetii was investigated. The use of 32P-labelled rickettsiae showed that phase II C. burnetii attached more readily to L cells than phase I organisms; this probably accounts for the more rapid establishment of infection of host cells by the phase II agents. Two lines of evidence indicated that C. burnetii plays a passive role in both attachment and entry into host cells: (1) inactivation of rickettsiae by either heat or glutaraldehyde did not affect either process, and (2) metabolic inhibitors of L cell phagocytic function--NaF and cytochalasin B and D--abolished rickettsiae uptake. These results indicate that it is an endocytotic event. While the presence of purified phase I lipopolysaccharide (LPS) did not interfere with attachment of rickettsiae to the surface of host cells, it markedly impaired entry of C. burnetii in both phases. This suggests that LPS is not an adhesin and that it is toxic to the host cell. Treatment of L cells with either pronase, subtilisin or subtilopeptidase A significantly reduced the number of C. burnetii that adhered to the host cell surface; this result suggests that proteins are either proximate to or components of the C. burnetii attachment site.

Animals↗
PubMed1996

Genetics of Coxiella burnetii.

Those organisms considered to be obligate intracellular bacteria are interesting objects for genetic studies. Little is known about their mechanisms for natural genetic exchange. Many genes from the bacterium Coxiella burnetii, an obligate intraphagolysosomal pathogen, have therefore been cloned and characterized using the heterologous host Escherichia coli. Recently, use of electroporation methodology followed by long-term selection periods have provided initial data on genetic transformation in C. burnetii.

Coxiella burnetii↗
PubMed2006

Goats may experience reproductive failures and shed Coxiella burnetii at two successive parturitions after a Q fever infection.

Q fever is a zoonosis caused by the obligate intracellular bacterium, Coxiella burnetii. Aborting domestic ruminants are the main source of human infection. In January 2003, an abortion episode occurred in a dairy caprine herd where 18/60 (30%) goats experienced reproductive problems: 4/60 (7%) aborted and 14/60 (23%) had stillbirths. Serological screening for abortion-related infectious diseases suggested Q fever. The diagnosis of C. burnetii infection was confirmed with PCR based on the occurrence of C. burnetii shedding into vaginal mucus, faeces and colostrums taken after kidding from the affected animals. The pregnancy following this episode resulted in one abortion and four stillbirths; three of those goats had already experienced reproductive failure during the previous kidding season. The seroprevalence of C. burnetii infection and the bacteria shedding were investigated using both ELISA and PCR assays, respectively, during the course of the initial and subsequent kidding seasons. Serological testing, performed on the whole herd 6 weeks after the abortion episode, showed 48/60 (80%) of ELISA positive goats. PCR assay performed on both vaginal swab and milk samples showed that the bacterium was shed for almost four months after the outbreak. C. burnetii DNA was also amplified from vaginal swab and milk samples taken from goats after the second kidding season. Furthermore, the bacteria were found into 14 vaginal swabs and 12 milk samples taken from infected females at both kidding seasons.

Abortion, Veterinary↗
PubMed1993

Seroepidemiology of Coxiella burnetii among wildlife in Nova Scotia.

We used an indirect immunofluorescence assay to determine antibody titers to phase I and phase II Coxiella burnetii antigens in serum samples from a variety of wild animals in Nova Scotia. Forty-nine percent of the hares, 16.5% of the moose, 7.1% of the raccoons, and 1.5% of the white-tailed deer tested had antibodies to phase I antigen. We conclude that there is extensive infection of the hare population by C. burnetii, with lesser degrees of infection of the moose, raccoon, and deer population.

Animals↗
PubMed2000

Comparison of a commercial enzyme-linked immunosorbent assay with immunofluorescence and complement fixation tests for detection of Coxiella burnetii (Q fever) immunoglobulin M.

A commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Q fever (PanBio Coxiella burnetii immunoglobulin M [IgM] ELISA, QFM-200) was compared to the indirect fluorescent antibody test (IFAT) for C. burnetii IgM and the complement fixation test (CFT). The ELISA demonstrated 92% agreement with the reference method (IFAT), and gave a sensitivity of 99% (69 of 70 samples) and a specificity of 88% (106 of 121). Specificity can be increased with confirmation by IFAT. CFT was found to have a specificity of 90% (107 of 119), although it was lacking in sensitivity (73%; 51 of 70). No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma (n = 6), chlamydia (n = 5), or legionella (n = 4) infections, although 2 of 5 patients with leptospirosis and 1 of 4 samples containing rheumatoid factor (RF) demonstrated positive results in the ELISA. Results indicate that the performance of the PanBio C. burnetii (Q fever) IgM ELISA (F = 187) is superior to that of CFT (F = 163), and consequently the ELISA should be a useful aid in the diagnosis of acute Q fever.

Antibodies, Bacterial↗
PubMed1979

Inhibition by Coxiella burnetii of ascites tumour formation in mice.

Mice injected intraperitoneally (i.p.) with killed purified Coxiella burnetii organisms were protected from ascites development and death caused by i.p. inoculation of sarcoma-180 cells. The extent of protection was a function of the relative dose of C. burnetii and tumour cells, and of the time of injection of C. burnetii. Phase II C. burnetii organisms exerted an antitumour protection at least as high as phase I C. burnetii organisms.

Animals↗
PubMed1986

Genetic heterogeneity among isolates of Coxiella burnetii.

Chromosomal and plasmid DNA have been extracted from six isolates of Coxiella burnetii, the aetiological agent of Q fever. Restriction fragment length polymorphisms detected after HaeIII digestions of chromosomal DNA revealed four different patterns that distinguished the American from the European isolates, and the Nine Mile phase I prototype strain from a spontaneously derived, isogenic phase II nonrevertant variant. At least one of the HaeIII fragments visible in the pattern from Nine Mile phase I and not in that from Nine Mile phase II could not be detected by DNA-DNA hybridization, and thus may have been deleted during the phase transition. Comparison of Nine Mile phase II, which does not survive animal passage, with Grita M44 phase II, which does, indicated that the HaeIII fragment was present in the Grita strain. These results suggest that this HaeIII fragment may be concerned with functions necessary to survive the cellular immune response in vivo. Isolates from two human endocarditis cases showed the greatest divergence from all the other isolates, having at least five fragments of unique mobility in the HaeIII digestion pattern of their chromosomal DNA. Also, a plasmid obtained from these two isolates was 2 to 3 kb larger than the plasmid present in the other five isolates, and its restriction pattern could be distinguished from that of the other plasmids by several endonucleases. Detection of chromosomal and plasmid restriction fragment length polymorphisms among strains of phase I or phase II C. burnetii from various geographical locations and environmental sources will facilitate Q fever diagnosis and strain identification.

Autoradiography↗
PubMed1998

PCR detection of Coxiella burnetii from different clinical specimens, especially bovine milk, on the basis of DNA preparation with a silica matrix.

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.

Animals↗
PubMed1979

Antibody response in man following a small intradermal inoculation with Coxiella burnetii phase I vaccine.

A small inoculum (0.2 microgram) of phase I Coxiella burnetii vaccine given to individuals previously sensitized to CO burnetii elicited a positive skin reaction and a strong IgM phase I antibody response as determined by microagglutination, complement fixation and microimmunofluorescence tests. A similar inoculum administered to nonsensitized individuals did not elitic a skin reaction nor stimulate a recognizable antibody response. Serum from one of these sensitized and skin tested individuals was fractionated by gel filtration methods. The serum and serum fractions were titrated in a mouse seroprotection test using primary chicken embryo cell culture plaque technique as the assay procedure. Results of the mouse seroprotection test indicated that most of the protective activity of the serum was associated with the IgM fraction and that phase I IgM antibody suppressed the growth of C. burnetii in mouse spleen when mixed with the rickettsial suspension prior to inoculation.

Adult↗
PubMed1999

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene.

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.

Animals↗
PubMedDate not supplied

[Coxiella burnetii infections and infections with bacteria of the genus Chlamydia in dairy cattle].

Comparative studies on the prevalence of infections caused by Coxiella burnetii (C. burnetii) and Chlamydia were carried out with 592 cattle older than 2 years and 234 cattle younger than 2 years. Of these 477 originated from 24 dairy herds with considerable fertility problems (positive herds) and 349 from 14 dairy herds without major fertility problems (control herds). For the direct detection of these pathogens in the genitals capture ELISAs were employed, for the demonstration of antibodies the complement fixation test (CFT). Direct detection of C. burnetii and Chlamydia single as well as mixed infection revealed significant higher values for cattle from positive herds compared with those from the control herds. Animals revealing insemination ratios of > or = 2 showed significantly more frequent excretion of Chlamydia via the genitals and antibodies against C. burnetii than cattle with an insemination ratio of < 2. Investigations of cows which had had an abortion showed no indications of significantly more frequent C. burnetii or chlamydial infections. Inseminated but non-pregnant cows excreted significantly more C. burnetii and Chlamydia than pregnant cows. Clinical signs of endometritis were associated with an enhanced excretion of Chlamydia. Animals younger than 2 years excreted significantly more frequently C. burnetii but not Chlamydia via the genitals than animals older than 2 years. Indirect test showed results vice versa.

Abortion, Veterinary↗
PubMed1989

[Properties of phase I purified cellular antigen of Coxiella burnetii].

An attempt was made to purify phase I cell suspension of Coxiella burnetii used as an antigen in diagnostic serological tests. Homogenised suspension of chick embryos infected with phase I Henzerling and "Z" strains, after preliminary purification from host cell contaminants of chick embryos was subjected to consecutive centrifugation in sucrose/uropoline gradient and to continuous 20-45% uropoline gradient. The fractions obtained from uropoline gradient centrifugation were applied as phase I antigen C. burnetii in the following tests: complement fixation and microagglutination. Only fractions containing protein were serologically active. They proved to be of similar specificity and sensitivity as the antigens obtained by standard method. Moreover, it was found that after formalin treatment of C. burnetii cells no soluble antigens are liberated which could be detected by complement fixation test.

Agglutination Tests↗
PubMed2004

Genetic control of natural resistance of mouse macrophages to Coxiella burnetii infection in vitro: macrophages from restrictive strains control parasitophorous vacuole maturation.

Macrophages from A/J and BALB/c mice were more susceptible to Coxiella burnetii phase II infection than were those from C57BL/6, C57BL/10, B10.A, C3H/HePas, and Swiss mice. Resistant macrophages effectively controlled the development of large replication vacuoles (LRVs), which accounted for the restriction of bacterial multiplication within the cultures. However, compared to fibroblasts, all macrophages controlled bacterial multiplication within LRVs.

Animals↗